The hedgehog signaling network regulates stem/progenitor cell function in many organs, including the mammary gland (1, 2). Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast, where ~80% of breast cancers show evidence of altered SMO-mediated signaling (2-4). However, the molecular and cellular mechanisms by which SMO functions in the breast, and perhaps in breast cancers, are not known. In vitro mammosphere-formation assays using human mammary epithelial cells suggest that canonical hedgehog signaling (via downstream activation of GLI transcription factors by SMO) stimulates both normal and malignant mammary stem/progenitor cell self-renewal (1). However, the requirement for SMO-mediated signaling in regenerative stem cells has not been demonstrated directly. Recent data from our laboratory using transgenic mice expressing activated human SMO (SmoM2) under the Mouse Mammary Tumor Virus (MMTV) promoter (MMTV-SmoM2) are consistent with the hypothesis that SMO-mediated hedgehog signaling can regulate mammary epithelial stem or progenitor cells in vivo (2). However, while MMTV-SmoM2 expression increased proliferation, proliferating cells did not express detectable SMO protein, nor was Gli mRNA expression increased as would be expected if canonical signaling was occurring. Similarly, in human breast cancers, the majority of cells expressing SMO were not proliferative. Recent groundbreaking in vitro studies demonstrate that SMO can function in non-canonical signaling and couple primarily with pertussis-toxin sensitive members of the Gi subfamily of alpha subunits of heterotrimeric G- proteins. Our own preliminary data now show that MMTV-SmoM2 can block pertussis toxin-induced proliferation in vivo. Together with other preliminary data, these results suggest the hypothesis that SMO- mediated hedgehog signaling is required for normal mammary stem/progenitor cell function. Further, we propose that SMO functions at least in part via a non-canonical mechanism as a G-protein-coupled receptor, and that it stimulates proliferation in a paracrine manner. To test this hypothesis, we propose four specific aims. In aim 1, we will test whether SMO is required for mammary epithelial stem/progenitor cell function using conditional loss- and gain-of-function alleles to evaluate stem cell behavior both in vivo and in vitro. In aim 2 we will test whether SMO activity can stimulate proliferation via a paracrine signaling mechanism in a series of in vitro cell culture assays, as well as in vivo using cell mixing transplantation assays, or two of our novel methods for generating chimeric mammary glands. In Aim 3 we will determine whether SMO functions via canonical GLI-mediated signaling, or via a novel non-canonical mechanism as a G-protein-coupled receptor in mouse mammary epithelial cells. Finally, in Aim 4, we will test whether SMO activation promotes breast cancer development induced by chemical carcinogens or by radiation. PROJECT NARRATIVE The requirement for SMO-mediated hedgehog signaling in stem/progenitor cell function has not been demonstrated directly, and the mechanism(s) by which ectopically expressed SMO functions to promote mammary hyperplasia (or breast cancer) are not clear. This project seeks to provide definitive data regarding the requirement for SMO in breast development, and to determine the extent to which SMO functions via canonical or non-canonical mechanisms. Given recent excitement about the possibility of using SMO antagonists to treat breast cancer patients, a full understanding of SMO function is absolutely essential prior to introduction of these agents into the clinic.